Quantifying a substance produced by a chemical reaction that occurs in biological specimens such as cells, cell aggregates, and pieces of tissue is a technique required for viability assay, functional assay and the like of biological specimens in fields such as medical and drug discovery. One method for quantifying a chemical reaction product released from a biological specimen is electrochemical measurement. For example, the progress of stem cell differentiation is monitored using electrochemical measurement in Non-patent literature 1.
Electrochemical measurement is a method in which an oxidization or reduction reaction is caused to a measurement target in an electrolytic solution in which two or more electrodes connected to an external power source are inserted, by removing electrons from the measurement target or supplying electrons to the measurement target through electrodes, while at the same time a current flowing between the electrodes is measured to determine whether an oxidation-reduction reaction has occurred, that is, to detect the presence or absence of the measurement target.
A typical electrochemical measurement apparatus includes a working electrode, which supplies or receives electrons to or from a measurement target to cause an oxidation-reduction reaction, a counter electrode, which is connected to the working electrode through an external power source and compensates for electron transfer occurring at the working electrode, an electrolytic solution, which enables transfer of electrons through ions in a measurement system and makes the entire measurement system a closed circuit, and a reference electrode for providing a reference for voltage.
In Non-patent literature 1, alkaline phosphatase (ALP), which is an undifferentiation marker and exists in the cell membranes of embryonic stem (ES) cells, is indirectly measured by electrochemical measurement for an embryoid body (EB) which is an aggregate of ES cells produced from ES cells of a mouse.
The reaction in which a stem cell whose function is yet to be determined changes to a somatic cell whose function is determined is commonly referred to as differentiation and a substance that indicates that differentiation has not occurred is referred to as an undifferentiation marker.
ALP is a cell undifferentiation marker and also has the property of hydrolyzing a phosphoric ester compound under alkaline conditions. For example, ALP acts as an enzyme in a reaction that changes p-aminophenyl phosphate (PAPP), which is a phosphoric ester compound, into p-aminophenol (PAP). PAP produced by the enzymatic reaction is a substance that is electrochemically active and is oxidized to p-quinone imine (PQI) by application of a voltage to the working electrode using the reference electrode as a reference. Specifically, the presence of ALP is detected as a current value in electrochemical measurement through two reactions of an enzymatic reaction and an oxidation-reduction reaction.
In Non-patent literature 1, a multielectrode amperometric device in which 20×20=400 working electrodes with a diameter of ϕ40 μm are provided in an array with a pitch of 250 μm is used for measurement. The device two-dimensionally images reactions in biological specimens of several to several hundred micrometers over time by using electrode current values acquired from the 400 electrodes.